野生茄子原生质体制备及电融合条件研究
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摘要
以野生茄子蒜芥茄(Solanum sisymbriifolium)和水茄(S. torvum)叶片为原生质体融合材料,对原生质体制备中酶解液浓度、酶解时间和电融合参数进行研究。结果显示:在0.3%(m/V)纤维素酶+0.1%(m/V)离析酶浓度条件下镜检观测到的单细胞数量最多;蒜芥茄酶解最适时间为14 h,原生质体产量达最大值15.06×10~6个·g~(-1),水茄酶解最适时间为13 h,原生质体产量达最大值15.92×10~6个·g~(-1);最适电融合参数为交流电场强度80 V·cm~(-1),作用时间10 s,直流脉冲强度1 250 V·cm~(-1),作用时间45μs,次数1次时,显微镜观测到融合效果最佳。
Taking the leaves of Salanum sisymbriifolium and S.torvum as protoplast fusion materials,this paper studied the enzymatic hydrolysate concentration,enzymatic hydrolysis time and electrofusion parameters.The results showed that the optimal concentration was 0.3%(m/V)cellulase R-10+0.1%(m/V)macerozyme R-10.Under this condition,the maximum number of single cells could be observed by microscope.The optimal enzymolysis time were 14 hours for S. sisymbriifolium and 13 hours for S. torvum,which the protoplasts yield was 14.06×10~6 protoplasts per gram and 15.92×10~6 protoplasts per gram,respectively.The optimum electric fusion parameters of field strength was 80V·cm~(-1),for 10 s action time.The D.C. square pulses was 1 250 V·cm~(-1) for 45 μs.Just once the best fusion effect could be observed by microscope.
Taking the leaves of Salanum sisymbriifolium and S.torvum as protoplast fusion materials,this paper studied the enzymatic hydrolysate concentration,enzymatic hydrolysis time and electrofusion parameters.The results showed that the optimal concentration was 0.3%(m/V)cellulase R-10+0.1%(m/V)macerozyme R-10.Under this condition,the maximum number of single cells could be observed by microscope.The optimal enzymolysis time were 14 hours for S. sisymbriifolium and 13 hours for S. torvum,which the protoplasts yield was 14.06×10~6 protoplasts per gram and 15.92×10~6 protoplasts per gram,respectively.The optimum electric fusion parameters of field strength was 80V·cm~(-1),for 10 s action time.The D.C. square pulses was 1 250 V·cm~(-1) for 45 μs.Just once the best fusion effect could be observed by microscope.
引文
常胜合,孙威,许桂莺,王展,银曼妮,韩谦,魏卿,李敬阳,舒海燕.2018.植物原生质体分离方法及其应用研究进展.分子植物育种,16(4):1271-1277.
郭文武,邓秀新,史永忠.1998.柑橘细胞电融合参数选择及种间体细胞杂种植株再生.植物学报,40(5):417-424.
田国奎,蔡兴奎,盛万民,李凤云,曹淑敏,牛志敏,李庆全,徐洪岩,娄树宝,王海艳.2012.二倍体马铃薯体细胞电融合参数的优化.遗传育种,26(6):321-324.
王爽,罗丰,肖春雷,刘勇,柯用春,孔祥义.2013.海南嫁接茄子死秧原因分析.中国蔬菜,(15):27-28.
喻艳.2013.马铃薯与茄子原生质体融合创制新资源研究[博士论文].武汉:华中农业大学.
张红浩,黄丽桃,韦慧明,赵曾菁,陈文明,阳燕娟,于文进.2015.茄子砧木种质资源的多样性及抗病性鉴定.南方农业学报,46(7):1232-1236.
Borgato L,Conicella C,Furini P A.2007.Production and characterization of arboreous and fertile Solanum melongena+Solanum marginatum somatic hybrid plants.Planta,226(4):961-969.
Daunay M C,Chaput M H,Sihachakr D,Allot M,Vedel F,Ducreux G.1993.Production and characterization of fertile somatic hybrids of eggplant(Solanum melongena L.)with Solanum aethiopicum L.Theor Appl Genet,85:841-850.
Daunay M C.2008.Eggplant//Prohens J,Nuez F.VegetablesⅡ.Handbook of Plant Breeding.New York:Springer.
Gleddie S,Keller W A,Setterfield G.1986.Production and characterization of somatic hybrids between Solanum melongena L.and S.sisymbriifolium Lam.Theor Appl Genet,71:613-621.
Guri A,Sink K C,1988.Interspecific somatic hybrid plants between eggplant(Solanum melongena)and Solanum torvum.Theor Appl Genet,76:490-496.
Jarl C I,Rietveld E M,Haas J M D.1999.Transfer of fungal tolerance through interspecific somatic hybridisation between solanum melongena and S.torvum.Plant Cell Reports,18(9):791-796.
Rotino G L,Sala T,Toppino L.2014.Eggplant//Pratap A,Kumar J.Alien gene transfer in crop plants.New York:Springer.
Sihachakr D,Haicour R,Serraf I,Barrientos E,Herbreteau C,Ducreux G,Rossignol L,Souvannavong V.1988.Electrofusion for the production of somatic hybrid plants of Solanum melongena L.and Solanum khasianum C.B.Clark.Plant Science,57(3):215-223.
Sihachakr D,Haicour R,Chaput M,Barrientos E,Ducreux G,Rossignol L.1989.Somatic hybrid plants produced by electrofusion between Solanum melongena L.and Solanum torvum Sw.Theoretical&Applied Genetics,77(1):1-6.321
Stattmann M,Gerick E,Wenzel G.1994.Interspecific somatic hybrids between Solanum khasianum and S.aculeatissimum produced by electrofusion.Plant Cell Reports,13(3-4):193-196.
郭文武,邓秀新,史永忠.1998.柑橘细胞电融合参数选择及种间体细胞杂种植株再生.植物学报,40(5):417-424.
田国奎,蔡兴奎,盛万民,李凤云,曹淑敏,牛志敏,李庆全,徐洪岩,娄树宝,王海艳.2012.二倍体马铃薯体细胞电融合参数的优化.遗传育种,26(6):321-324.
王爽,罗丰,肖春雷,刘勇,柯用春,孔祥义.2013.海南嫁接茄子死秧原因分析.中国蔬菜,(15):27-28.
喻艳.2013.马铃薯与茄子原生质体融合创制新资源研究[博士论文].武汉:华中农业大学.
张红浩,黄丽桃,韦慧明,赵曾菁,陈文明,阳燕娟,于文进.2015.茄子砧木种质资源的多样性及抗病性鉴定.南方农业学报,46(7):1232-1236.
Borgato L,Conicella C,Furini P A.2007.Production and characterization of arboreous and fertile Solanum melongena+Solanum marginatum somatic hybrid plants.Planta,226(4):961-969.
Daunay M C,Chaput M H,Sihachakr D,Allot M,Vedel F,Ducreux G.1993.Production and characterization of fertile somatic hybrids of eggplant(Solanum melongena L.)with Solanum aethiopicum L.Theor Appl Genet,85:841-850.
Daunay M C.2008.Eggplant//Prohens J,Nuez F.VegetablesⅡ.Handbook of Plant Breeding.New York:Springer.
Gleddie S,Keller W A,Setterfield G.1986.Production and characterization of somatic hybrids between Solanum melongena L.and S.sisymbriifolium Lam.Theor Appl Genet,71:613-621.
Guri A,Sink K C,1988.Interspecific somatic hybrid plants between eggplant(Solanum melongena)and Solanum torvum.Theor Appl Genet,76:490-496.
Jarl C I,Rietveld E M,Haas J M D.1999.Transfer of fungal tolerance through interspecific somatic hybridisation between solanum melongena and S.torvum.Plant Cell Reports,18(9):791-796.
Rotino G L,Sala T,Toppino L.2014.Eggplant//Pratap A,Kumar J.Alien gene transfer in crop plants.New York:Springer.
Sihachakr D,Haicour R,Serraf I,Barrientos E,Herbreteau C,Ducreux G,Rossignol L,Souvannavong V.1988.Electrofusion for the production of somatic hybrid plants of Solanum melongena L.and Solanum khasianum C.B.Clark.Plant Science,57(3):215-223.
Sihachakr D,Haicour R,Chaput M,Barrientos E,Ducreux G,Rossignol L.1989.Somatic hybrid plants produced by electrofusion between Solanum melongena L.and Solanum torvum Sw.Theoretical&Applied Genetics,77(1):1-6.321
Stattmann M,Gerick E,Wenzel G.1994.Interspecific somatic hybrids between Solanum khasianum and S.aculeatissimum produced by electrofusion.Plant Cell Reports,13(3-4):193-196.